Gel-matrix electrophoresis

ABSTRACT

The invention provides an electrophoresis gel-matrix layer, usually adhered upon a support plate, having two mutually opposite ends for application of an electrophoresis voltage thereto, an exposed major surface extending between the two ends, and a plurality of wells in the thickness of the layer and open at the said exposed surface, wherein the wells are arranged in a plurality of rows each extending transversely of the end-to-end direction of the layer and the wells in successive rows are progressively offset in the transverse direction whereby electrophoresis tracks obtained from wells of one row will pass, if extended so far, between wells of at least one other row and tracks obtained therefrom. The invention also provides a method of making, and a method of using, such a gel-matrix layer.

This application is a National Stage of International Application No.PCT/GB94/02745, filed Dec. 15, 1994.

FIELD OF THE INVENTION

This invention relates to electrophoresis, in particular theelectrophoresis of numerous samples.

BACKGROUND OF THE INVENTION

Electrophoresis of, for example, samples of DNA fragments are commonlyperformed in a gel matrix of either agarose or acrylamide. In the caseof agarose, the gel is usually poured while molten on to a horizontalglass plate on which it forms a layer which then sets; and in order tohandle a plurality of samples simultaneously a "comb" having a row ofprotruding teeth or pegs is positioned so that the pegs project into theagarose layer while it sets. When the agarose layer has set, the comb isremoved to leave a row of holes or wells in the layer adjacent one edgeof the plate. These wells can then be loaded with samples which are thensubjected simultaneously to electrophoresis by applying a voltage fromend to end of the agarose layer so as to form a corresponding number ofparallel electrophoresis tracks each extending from a respective one ofthe loaded wells towards the other end of the plate. In order toaccommodate a larger number of samples than can conveniently be loadedinto a single row of wells, the comb may be formed with a second row ofpegs to form a second row of wells in the agarose, the second row beingspaced from the first, and also from the other end of the plate, by adistance greater than the desired length of the electrophoresis trackswhich are to be obtained.

In the case of an acrylamide gel layer, the open-faced preparationmethod used for agarose is not suitable, because acrylamide does notpolymerise in the presence of air. It is therefore usual to prepare theacrylamide as a sandwich layer between two glass plates and, sinceneither major face of the layer is accessible, to form the wells in oneend edge of the acrylamide layer. Electrophoresis is then carried outwith the sandwich vertical, and its edge in which the wells are formedas its upper edge, so that samples can be loaded into the wells. Itwould not be practical in such a case to provide a spaced additional rowof wells to enable a larger number of samples to be handledsimultaneously. Acrylamide does, however, provide higher resolution ofsamples and, other considerations being equal, would be preferred toagarose.

There is a need for an electrophoresis gel-matrix layer which enableslarger numbers of samples to be handled simultaneously than is possiblewith either of the known multiple-well plates described above, and it isan object of the invention to provide an improved electrophoresisgel-matrix layer which achieves this.

SUMMARY OF THE INVENTION

According to the invention there is provided an electrophoresisgel-matrix layer having two mutually opposite ends for application of anelectrophoresis voltage thereto, an exposed major surface extendingbetween the two ends, and a plurality of wells in the thickness of thelayer and open at the said exposed surface, wherein the wells arearranged in a plurality of rows each extending transversely of theend-to-end direction of the layer and the wells in successive rows areprogressively offset in the transverse direction whereby electrophoresistracks obtained from wells of one row will pass, if extended so far,between wells of at least one other row and tracks obtained therefrom.

The invention also provides a method of making, and a method of using,such a gel-matrix layer.

Usually, the gel-matrix layer according to the invention will be adheredupon a support plate.

Usually also, the wells will be arranged in a regular lattice. In apreferred embodiment of the invention, the wells are arranged in asquare lattice pattern of equispaced rows and columns, perpendicular toone another and both offset at an angle to the end-to-end direction ofthe matrix layer. If for example, the transverse rows of wells are at anangle of tan⁻¹ 3 (about 711/2°) to the end-to-end direction of thelayer, or at 181/2° to the ends of the layer, electrophoresis tracksoriginating at the wells of one row will pass between wells of the nexttwo rows, before (if they extend so far) colliding with wells in thenext row encountered.

The invention, and preferred embodiments of electrophoretic gel-matrixlayers and plates in accordance with it, are disclosed in greater detailin the following description with reference to the accompanyingdrawings, immediately

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a toothed or pegged mould for casting agel-matrix layer;

FIG. 2 is a side view of the mould shown in FIG. 1;

FIG. 3 is a view similar to that shown in FIG. 2, but partly broken awayand showing a gel layer within the mould and a support plate laid overthe gel layer;

FIG. 4 is a plan view of the gel layer after it and its support platehave been removed from the mould; and

FIG. 5 is an end view of the gel layer shown in FIG. 4 and of itssupport plate.

DETAILED DESCRIPTION

The mould shown in FIG. 1 comprises a rectangular base plate 11 havingends 12, sides 13 and a flat upper surface 14 from which project aplurality of pegs or teeth 15. The teeth 15 are disposed in a regularsquare lattice pattern, in rows and columns which are angularly offsetrelative to the ends 12, or the end-to-end direction, of the mould 11.As illustrated (and as will be seen more clearly from FIG. 4 referred tobelow) the rows of teeth 15, extending transversely across the surface14 of the mould, are at an angle of tan⁻¹ 3 (about 711/2°) to the sides13 or the end-to-end direction of the mould and thus at about 181/2° tothe ends 12.

Preferably, and as shown, there are twelve rows of teeth 15, with eightteeth in each row, and the centre-to-centre spacing of the teeth in bothdirections is 9 mm. Preferably also, each tooth is of squarecross-section, with its side faces parallel to the ends 12 and sides 13of the mould 11, with cross-sectional dimensions of 2 mm×2 mm and also aheight of 2 mm. The plate 11 and teeth 15 may be formed integrally froma single block of, for example, Perspex, by machining away part of thethickness of the block (except where the teeth are left) so as to leavethe teeth upstanding from the remainder of the thickness.

Round its perimeter, the mould 11 is provided with a rim 16 which isclosely adherent to the ends 12 and sides 13 and projects upwardly fromthem by an amount, say 3 mm, which is greater than the height of theteeth 15.

In use of the mould 11 with acrylamide to form the gel layer, it isfilled, to a depth at which the tops of the teeth 15 are just covered,with the acrylamide material 17 which is to polymerise to form thedesired gel-matrix layer; and a cover plate 18, as shown in FIG. 3, iscarefully laid on the material 17, progressively from one end to theother, to avoid entrapment of any air as bubbles between the material 17and the plate 18, and is pressed down on to the teeth 15 so that nomaterial 17 remains between the teeth 15 and the plate 18. Any excess ofthe material 17 is expelled over the rim 16, and the remaining material17 is allowed to polymerise and to become firmly adherent to the plate18. The acrylic material 17 is then polymerised to polyacrylamide,suitably by TEMED and ammonium persulphate. If the material 17 isacrylamide, that surface of the plate 18 to which it is to adhere ispreferably pretreated by silanising with a silanising agent which maysuitably be 0.5% gamma-methacryloxypropyltrimethooxysilane/0.5% glacialacetic acid/ethanol v/v, as obtainable from Sigma Chemicals.

If the layer 17 is to be of agarose gel, it is poured in at atemperature at which it is suitably liquid, but spacers (notillustrated) are first laid on the surface 14 to prevent the plate 18from resting on the teeth 15 and to ensure that agarose material remainsin the gaps between the teeth and the plate 18.

When the material 17 has polymerised or set and is firmly adhered to theplate 18, both are removed, together, from the mould. This can beachieved by carefully prising one end of the plate 18 away from thesurface 14 of the mould, when the polymerised gel-matrix layer 17remains adherent to the plate 18 and peels away from the mould surface14 and teeth 15. The resulting gel-matrix electrophoresis plate 19,consisting of the layer 17 and the support plate 18 to which it isadhered, is shown in FIGS. 4 and 5. Formed in the gel layer 17 are wells20, complementary to the teeth 15. If the layer 17 is acrylamide, theplate 18 forms the bottom of the wells 20, and the bond between the gellayer and the plate at their interface is effective to prevent sampleleakage along the interface from the bottoms of the wells. If the gellayer is of agarose, where the interface bonding would be less effectiveto prevent leakage, the bottoms of the wells are formed by the agarosewhich was left between the plate 18 and the teeth 15 by providing theabove-mentioned spacers for the plate 18. The wells 20, like the teeth15, are arranged on a square lattice of twelve rows, each of eightwells, with the rows, extending transversely across the width of theplate 19, disposed at an angle α to the lengthwise direction of theplate. As illustrated, though only by way of example, the angle α issuch that tanα=3. This means that when a sample loaded into a well 20 issubjected to electrophoresis by applying a voltage between ends 21 ofthe layer 17 so as to provide in the layer 17 an electric field in theend-to-end direction parallel to sides 22 of the plate, the resultingelectrophoresis track 23, extending in the lengthwise direction parallelto the sides 22, can pass between wells of the next two rows of wellswithout interfering either with them or with tracks originating withthem, before encountering one of the wells in the third row.

It will be seen that this embodiment of the invention, and the inventiongenerally, provides a remarkably compact arrangement of wells and acorrespondingly efficient use of the available gel. Furthermore, theparticular arrangement shown in FIG. 4, namely the arrangement of thewells 20 in a 12×8 square lattice pattern with 9 mm centre-to-centrespacing, is the same as that of the wells of a standard microtitreplate. It will very often be the case that the samples which are to beanalysed or examined by electrophoresis will have been produced, inlarge numbers, in standard microtitre plates; and the use of anelectrophoresis plate like that in FIG. 4, with its wells arranged inthe identical manner, can lead to great economies of time and laboursince the transfer of samples from the wells of a microtitre plate tothe wells of the electrophoresis plate can be performed automatically orsemi-automatically by the use of a multichannel pipette.

It is with this use of multichannel pipettes, possibly roboticallyoperated, in mind that the wells 20 are preferably made not much smallerthan 2 mm×2 mm, which allows for some misalignment of individual pipettetips. As stated above, with wells of these dimensions in a 9 mm×9 mmlattice it is permissible to choose an angular offset such that α=tan⁻¹3. If an even more compact arrangement were required, in which (stillwith the 9 mm×9 mm lattice spacing) the track 23 from a well in one rowwould pass through the next three rows without interference beforeencountering another well in the fourth row, thus requiring reducedangular offset such that tan α=4, it would probably be advisable toreduce the transverse dimension of the tracks 23, and thus of the wells20, from 2 mm to about 1.5 mm. The maximum track length when tan α=3 isabout 28 mm, which is adequate, when using polyacrylamide as the gellayer 17, for many pattern recognition analyses which depend on mobilitydifferences greater than 5-10%. Increasing a to a value such that tan a=4 gives an increased available track length of about 35 mm.

A further advantage of arranging the wells 20 identically with those ofa standard microtitre plate is that the same standard microtitre gridtransparency which has been marked up to identify the samples in thewells of a microtitre plate can also be used to identify the tracks 23obtained from those samples after they have been transferred to thecorresponding wells an electrophoresis plate 19. After electrophoresisand any necessary staining or other procedure to reveal the tracks 23,the marked-up microtitre grid transparency is used as an overlay on theplate 19 while it is photographed, to provide a record identifying thetracks with maximum simplicity and minimum expenditure of time on thenecessary record keeping.

As shown in FIG. 4, the plate 19 has a well-free region at its lowerend, to accommodate tracks 23 from the adjacent wells 20. A similarplate, if it also has a well-free region to one side of its array ofwells 20, may be re-used by turning it. through 90°, i.e. by subjectingit to an electrophoresis voltage inducing a field from side to side ofthe plate as viewed in FIG. 4. The tracks resulting from the second usewill be at right angles to the tracks 23 from the first use, and canreadily be distinguished and read without confusion.

What is claimed is:
 1. An electrophoresis gel-matrix layer having twomutually opposite ends for application of an electrophoresis voltagethereto, an exposed major surface extending between the two ends, and aplurality of wells in the thickness of the layer and open at the saidexposed surface, wherein the wells are arranged in a plurality of rowseach extending transversely of the end-to-end direction of the layer andthe wells in successive rows are progressively offset in the transversedirection whereby electrophoresis tracks obtained from wells of one rowwill pass, if extended so far, between wells of at least one other rowand tracks obtained therefrom.
 2. A gel-matrix layer as claimed in claim1, wherein the wells are arranged in a regular lattice pattern.
 3. Agel-matrix layer as claimed in claim 2, wherein the wells are arrangedin a square lattice pattern of equispaced transverse rows and columnswhich are perpendicular to the rows, and the rows and columns areangularly offset at respective angles relative to the end-to-enddirection of the matrix layer.
 4. A gel-matrix layer as claimed in claim3, wherein the rows of wells are disposed at an angle of about tan⁻¹ 3to the end-to-end direction of the layer.
 5. A gel-matrix layer asclaimed in claim 3, wherein the rows of wells are disposed at an angleof about tan⁻¹ 4 to the end-to-end section of the layer.
 6. A gel-matrixlayer as claimed in claim 1, supported by and adhered upon a supportplate.
 7. A gel-matrix layer adhered to a support plate, as claimed inclaim 6, wherein the gel-matrix layer is of agarose.
 8. A gel-matrixlayer adhered to a support plate, as claimed in claim 6, wherein thegel-matrix layer is of polyacrylamide.
 9. A method of making anelectrophoresis gel-matrix layer adhered upon a support plate,comprising the steps of:providing a rectangular mould tray having ahorizontally disposed flat surface bounded by upstanding side walls andend walls and having a plurality of pegs or teeth projecting upwardlyfrom the flat surface, introducing gel-forming material in flowablecondition into the mould to a depth at which it covers the pegs orteeth, laying a support plate on the gel-forming material in the mould,in contact with the exposed free surface of said material, allowing thegel-forming material to gel and adhere to the support plate, andremoving the support layer, and the gel layer adhered thereto, togetherfrom the mould and the pegs or teeth thereof, wherein the pegs or teethare arranged in a plurality of rows each extending across the said flatsurface of the mould transversely of the end-to-end direction of thesaid flat surface, with the pegs or teeth in successive rows beingprogressively offset in the transverse direction.
 10. A method asclaimed in claim 9, wherein the pegs or teeth of the mould are arrangedin a regular lattice pattern.
 11. A method of effecting simultaneouselectrophoresis of multiple samples, comprising the steps ofplacing eachsample in a respective one of a plurality of wells arranged in a latticepattern of rows and columns in a layer of gel material, and subjectingall the samples to electrophoresis by applying a voltage in a direction,along the layer of gel material, which is at an angle to the rows ofwells such that an electrophoresis track extending in that directionfrom a well in one of said rows will pass between wells in at least oneadjacent row before encountering, if sufficiently extended, a well in amore remote row.
 12. A method as claimed in claim 11 and including thepreliminary step of removing the samples, simultaneously, each from arespective one of a plurality of wells of a microtitre plate, prior toplacing the samples simultaneously in the wells in the layer of gelmaterial, the wells of the microtitre plate being arranged in the samelattice pattern of rows and columns as that of the wells of the layer ofgel material.